Preferential production and secretion of the complement regulator factor H-like protein 1 (FHL-1) by human myeloid cells.

Factor H is a pivotal complement regulatory protein that is preferentially produced by the liver and circulates in high concentrations in serum. There has been an increasing interest in the extrahepatic production of complement factors, including by cells of the immune system, since this contributes to non-canonical functions of local complement activation and regulation. Here we investigated the production and regulation of factor H and its splice variant factor H-like protein 1 (FHL-1) by human myeloid cells. As validation, we confirmed the predominant presence of intact factor H in serum, despite a strong but comparable mRNA expression of CFH and FHL1 in liver. Comparable levels of CFH and FHL1 were also observed in renal tissue, although a dominant staining for FHL-1 was shown within the proximal tubules. Human in vitro generated pro- and anti-inflammatory macrophages both expressed and produced factor H/FHL-1, but this was strongest in pro-inflammatory macrophages. Production was not affected by LPS activation, but was increased upon stimulation with IFN-γ or CD40L. Importantly, in both macrophage subsets mRNA expression of FHL1 was significantly higher than CFH. Moreover, production of FHL-1 protein could be confirmed using precipitation and immunoblotting of culture supernatants. These data identify macrophages as producers of factor H and FHL-1, thereby potentially contributing to local complement regulation at sites of inflammation.

Properdin produced by dendritic cells contributes to the activation of T cells.

The complement system does not only play an important role in the defence against microorganism and pathogens, but also contributes to the regulation of innate and adaptive immunity. Especially activation fragments C3a and C5a and complement activation at the interface of antigen presenting cell (APC) and T cell, were shown to have a role in T cell activation and proliferation. Whereas most complement factors are produced by the liver, properdin, a positive regulator of the C3 convertase, is mainly produced by myeloid cells. Here we show that properdin can be detected in myeloid cell infiltrate during human renal allograft rejection. In vitro, properdin is produced and secreted by human immature dendritic cells (iDCs), which is further increased by CD40-L-matured DCs (mDCs). Transfection with a specific properdin siRNA reduced properdin secretion by iDCs and mDCs, without affecting the expression of co-stimulatory markers CD80 and CD86. Co-culture of properdin siRNA-transfected iDCs and mDCs with human allogeneic T cells resulted in reduced T cell proliferation, especially under lower DC-T cell ratio's (1:30 and 1:90 ratio). In addition, T cell cytokines were altered, including a reduced TNF-α and IL-17 secretion by T cells co-cultured with properdin siRNA-transfected iDCs. Taken together, these results indicate a local role for properdin during the interaction of DCs and allogeneic T cells, contributing to the shaping of T cell proliferation and activation.

Initial properdin binding contributes to alternative pathway activation at the surface of viable and necrotic cells.

Properdin, the only known positive regulator of the complement system, stabilizes the C3 convertase, thereby increasing its half-life. In contrast to most other complement factors, properdin is mainly produced extrahepatically by myeloid cells. Recent data suggest a role for properdin as a pattern recognition molecule. Here, we confirmed previous findings of properdin binding to different necrotic cells including Jurkat T cells. Binding can occur independent of C3, as demonstrated by HAP-1 C3 KO cells, excluding a role for endogenous C3. In view of the cellular source of properdin, interaction with myeloid cells was examined. Properdin bound to the surface of viable monocyte-derived pro- and anti-inflammatory macrophages, but not to DCs. Binding was demonstrated for purified properdin as well as fractionated P2, P3, and P4 properdin oligomers. Binding contributed to local complement activation as determined by C3 and C5b-9 deposition on the cell surfaces and seems a prerequisite for alternative pathway activation. Interaction of properdin with cell surfaces could be inhibited with the tick protein Salp20 and by different polysaccharides, depending on sulfation and chain length. These data identify properdin as a factor interacting with different cell surfaces, being either dead or alive, contributing to the local stimulation of complement activation.

Culture medium used during small interfering RNA (siRNA) transfection determines the maturation status of dendritic cells.

Gene silencing using small interfering ribonucleic acids (siRNA) is a powerful method to interfere with gene expression, allowing for the functional exploration of specific genes. siRNA interference can be applied in both cell lines, as well as in primary, non-dividing cell types like dendritic cells. However, the efficacy in different cell types is variable and requires optimization. Here, we showed that the type of culture medium used during lipid-based siRNA-mediated transfection acts as a critical factor, affecting dendritic cell activation. Transfection of immature monocyte-derived dendritic cells in RPMI medium, but not in IMDM, showed increased transcript levels of pro-inflammatory cytokines. Moreover, the expression of co-stimulatory molecules was enhanced, thereby increasing the T cell stimulatory capacity. Our data demonstrates that the choice of medium should be critically examined as one of the variables while optimizing cell transfection.

Identification of a novel non-coding mutation in C1qB in a Dutch child with C1q deficiency associated with recurrent infections.

INTRODUCTION: C1q deficiency is a rare genetic disorder that is strongly associated with development of systemic lupus erythematosus (SLE). Several mutations in the coding regions of the C1q genes have been described that result in stop-codons or other genetic abnormalities ultimately leading to C1q deficiency. Here we report on a Dutch boy suffering from recurrent infections with a complete C1q deficiency, without any SLE symptoms. METHODS: The presence of C1q in serum was assessed using ELISA and hemolytic assay. By western blot we examined the different C1q chains in cell lysates. We identified the mutation using deep-sequencing. By qPCR we studied the mRNA expression of C1qA, C1qB and C1qC in the PBMCs of the patient. RESULTS: Deep-sequencing revealed a homozygous mutation in the non-coding region of C1qB in the patient, whereas both parents were heterozygous. The mutation is located two nucleotides before the splice site of the second exon. In-silico analyses predict a complete abrogation of this natural splice site. Analyses of in vitro cultured cells from the patient revealed a lack of production of C1q and intracellular absence of C1qB in the presence of C1qA and C1qC peptides. Quantitative PCR analysis revealed total absence of C1qB mRNA, a reduced level of C1qA mRNA and normal levels of C1qC mRNA. CONCLUSION: In this study we report a new mutation in the non-coding region of C1qB that is associated with C1q deficiency.

Differential glycosylation of polymeric and monomeric IgA: a possible role in glomerular inflammation in IgA nephropathy.

IgA nephropathy (IgAN) is characterized by mesangial deposition of polymeric IgA1 (pIgA1) and complement. Complement activation via mannose-binding lectin and the lectin pathway is associated with disease progression. Furthermore, recent studies have indicated a possible role for secretory IgA. IgAN is associated with abnormalities in circulating IgA, including aberrant O-linked glycosylation. This study characterized and compared functional properties and N-linked glycosylation of highly purified monomeric IgA (mIgA) and pIgA from patients with IgAN and control subjects. Total serum IgA was affinity-purified from patients (n = 11) and control subjects (n = 11) followed by size separation. pIgA but not mIgA contained secretory IgA, and its concentration was significantly higher in patients with IgAN than in control subjects. Both in patients with IgAN and in control subjects, IgA binding to the GalNAc-specific lectin Helix Aspersa and to mannose-binding lectin was much stronger for pIgA than for mIgA. Furthermore, binding of IgA to mesangial cells largely was restricted to polymeric IgA. Binding of pIgA to mesangial cells resulted in increased production of IL-8, predominantly with IgA from patients with IgAN. Quantitative analysis of N-linked glycosylation of IgA heavy chains showed significant differences in glycan composition between mIgA and pIgA, including the presence of oligomannose exclusively on pIgA. In conclusion, binding and activation of mesangial cells, as well as lectin pathway activation, is a predominant characteristic of pIgA as opposed to mIgA. Furthermore, pIgA has different N-glycans, which may recruit lectins of the inflammatory pathway. These results underscore the role of pIgA in glomerular inflammation in IgAN.

Glomerular activation of the lectin pathway of complement in IgA nephropathy is associated with more severe renal disease.

IgA nephropathy (IgAN) is characterized by glomerular co-deposition of IgA and complement components. Earlier studies showed that IgA activates the alternative pathway of complement, whereas more recent data also indicate activation of the lectin pathway. The lectin pathway can be activated by binding of mannose-binding lectin (MBL) and ficolins to carbohydrate ligands, followed by activation of MBL-associated serine proteases and C4. This study examined the potential role of the lectin pathway in IgAN. Renal biopsies of patients with IgAN (n=60) showed mesangial deposition of IgA1 but not IgA2. Glomerular deposition of MBL was observed in 15 (25%) of 60 cases with IgAN and showed a mesangial pattern. All MBL-positive case, but none of the MBL-negative cases showed glomerular co-deposition of L-ficolin, MBL-associated serine proteases, and C4d. Glomerular deposition of MBL and L-ficolin was associated with more pronounced histologic damage, as evidenced by increased mesangial proliferation, extracapillary proliferation, glomerular sclerosis, and interstitial infiltration, as well as with significantly more proteinuria. Patients who had IgAN with or without glomerular MBL deposition did not show significant differences in serum levels of MBL, L-ficolin, or IgA or in the size distribution of circulating IgA. Furthermore, in vitro experiments showed clear binding of MBL to polymeric but not monomeric patient IgA, without a significant difference between both groups. Together, these findings strongly point to a role for the lectin pathway of complement in glomerular complement activation in IgAN and suggest a contribution for both MBL and L-ficolin in the progression of the disease.

T-cell-specific immunosuppression results in more than 53 days survival of porcine islets of langerhans in the monkey.

BACKGROUND: Transplantation of islets of Langerhans can restore insulin production in diabetic patients. Because of the shortage of human donor organs, transplantation of porcine islets may be an alternative solution. The present study was aimed at the characterization of rejection mechanisms of porcine islets transplanted into eight nondiabetic monkeys under the kidney capsule. METHODS: Cultured adult pig islets were used, which showed no expression of the galactose(alpha1,3)galactose epitope, major histocompatibility complex class II, or CD45, and no binding of antibodies or complement after exposure to monkey serum. Immunosuppression consisted of cyclophosphamide, cyclosporine A (CsA), and steroids (group 1); or antithymocyte globulin, anti-interleukin-2 receptor antibody, CsA, and steroids (group 2). In three animals of group 2, islets were also transplanted in the portal vein. RESULTS: Although all monkeys had preformed anti-pig antibodies, no correlation was found between antibody titers and rejection and no deposition of antibodies or complement was observed in the grafts. Group 1 showed islets up to day 11, followed by T-cell infiltration and rejection at approximately day 14. In group 2, two monkeys showed infiltrates consisting predominantly of T cells starting at approximately day 29, whereas two monkeys showed well-preserved islets without infiltration up to day 53. In the livers of the three monkeys that also received islets intraportally and were resectioned on days 21, 33, and 49, no islets could be detected. CONCLUSIONS: This study demonstrates that cultured adult pig islets can survive in the monkey for more than 53 days without signs of rejection under standard immunosuppression.